This invention relates to separation of individual solutes from a mixture. More specifically, the invention relates to exploitation of carrier mediated transport mechanisms in immobilized liquid membrane and related separation systems.
There is a continuing need for more efficient methods of separating the solutes in mixtures of structurally related bioactive compounds. Synthesis of bioactive materials such as pharmaceuticals and pesticides using organic chemistries often requires separations on a preparative scale. The products of expression of engineered microorganisms and cell lines typically comprise a single valuable species in admixture with a host of extraneous proteins, lipids, nucleic acids, and polysaccharides. Isolation of pure or substantially pure product on a commercial scale from such mixtures presents a significant engineering challenge. Chromatography, differential precipitation, filtration, and other separation technologies are used to remove selectively unwanted species. Often, the late-purification stages are most difficult and can be achieved only by means of affinity chromatography using polyclonal or monoclonal antibodies which selectively immunochemically bind with the product.
Molecular biology has advanced as a technology sufficient to permit production of a variety of valuable biologically active proteins Pending U.S. application Ser. Nos. 052,800 and 342,449, and PCT Application US88/01737, disclose biosynthetic multifunctional proteins comprising plural bioactive domains, one of which is capable of binding to a preselected compound. The binding domain is patterned after the antibody binding site and can mimic the binding properties of a light or heavy chain protein, or comprises a single chain construct comprising both light and heavy chains. Such constructs may be used in affinity chromatography procedures and may have significant cost advantages over monoclonal antibodies.
Among the most difficult purification tasks is the separation of stereoisomers. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane polarized light. In describing an optically active compound, the prefixes D and L, or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes designate the sign of rotation of plane polarized light by the compound, with (-) or L meaning that the compound is levorotatory. For a given chemical structure, D and L stereoisomers are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric or racemic mixture. The term "racemic mixture" as used herein, refers to a mixture of at least first and second stereoisomers in any proportion.
Optical activity is typically the result of molecular asymmetry about tetrahedral carbon atoms that are linked to four different moieties. Where there is only one asymmetric carbon atom, or "chiral center", there are two possible stereoisomers or enantiomers. Where there are n chiral centers, the number of potential stereoisomers increases to 2.sup.n. The structural differences between stereoisomers are subtle and of little consequence in ordinary chemical reactions, and this accounts for many of the difficulties encountered in resolving racemic mixtures. However, these small structural differences may be profound in biological systems, e.g., if the compounds are involved in enzyme-catalyzed reactions or bind specifically to cellular receptors. Thus, the L-amino acids are metabolized in humans but the corresponding D analogs are not. Only D-glucose can be phosphorylated and processed into glycogen or degraded by the glycolytic and oxidative pathways of intermediary metabolism. Similarly, beta blockers, pheromones, prostaglandins, steroids, flavoring and fragrance agents, pharmaceuticals, pesticides, herbicides, and many other compounds exhibit critical stereospecificity. In the field of pesticides, for example, Tessier has shown that only two of the eight stereoisomers of deltamethrin, a pyrethroid insecticide, have any biological activity. (Chemistry and Industry, Mar. 19, 1984, p. 199). Other forms of optical isomers are known which are of commercial interest.
Stereochemical purity is also important in the field of pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit chirality. A case in point is provided by naproxen, or (+) S-2-(6-methoxy-2-naphthyl) propionic acid, used for instance in the management of arthritis. In this case, the S(+) enantiomer of the drug is known to be 28 times more therapeutically potent than its R(-) counterpart. Still another example of chiral pharmaceuticals is provided by the family of beta-blockers; the L-form of propranolol is known to be 100 times more potent than the D-enantiomer.
Synthesis of chiral compounds by standard organic synthetic techniques generally leads to a racemic mixture which, in the aggregate, may have a relatively low specific bioactivity since certain of the stereoisomers in the mixture are likely to be biologically or functionally inactive. As a result, relatively larger quantities of the material must be used to obtain an effective dose, and manufacturing costs are increased due to the co-production of stereochemically "incorrect" and hence, inactive ingredients. Also, the inactive enantiomer may have unwanted side effects.
A widely used approach to purifying optical isomers is the selective precipitation of the desired compound from a racemic mixture. See, for example, U.S. Pat. No. 3,879,451, 4,257,976, 4,151,198, 4,454,344; Harrison et al, J. Med. Chem. 13:203 (1970); Felder et al, UK Patent Application No. GB2025968A (1980), and U.S. 4,285,884. Separation of diastereomers also can be carried out by chromatography. See, for example, Pollock et al, J. Gas Chromatogr. 3:174 (1965); Mikes et al, J. Chromatogr. 112:205 (1976); and Hare et al, U.S. Pat. No. 4,290,893. Enzymes have been used for the resolution of stereoisomers on a preparative scale. For instance, enzymatic treatment has been applied to the resolution of racemic mixtures of amino esters. See U.S. Pat. No. 3,963,573, U.S. Pat. No. 4,262,092, Clement and Porter, J. Chem. Ed., 48:695 (1971), and Matta et al J. Org. Chem., 39:2291 (1974). Additional examples of enzyme-mediated resolution as applied to the production of optically purified pharmaceuticals include Sih U.S. Pat. No. 4,584,370, Aragozzini et al, Biotechnol Letters, 8:95 (1980), Yokozeki et al, EPO No. 0 122 794 A2, and Sih, Tetrahedron Letters, 27:1763 (1986). Multiphase and extractive enzyme membrane bioreactors that selectively produce pure or substantially purified optically active compounds from achiral precursors or mixtures are disclosed by Matson in U.S. Pat. No. 4,800,162. The term "resolution" as used herein, refers to separation of a first mixture into second and third mixtures wherein the proportions of the solutes in the second and third mixtures are different from that in the first mixture, the proportion being greater in one and necessarily smaller in the other.
Permeation of solutes through liquids has been exploited for separation of certain ions using an immiscible liquid film as a membrane to mediate transport. In these so-called "immobilized liquid membranes", a microporous solid support holds the liquid as a continuous barrier between the feed and product streams. The liquid is held in place by capillarity and assumes the geometry of the support. A novel variation of this technology exploits coupled transport. An extracting agent in the membrane selectively complexes a metal ion in the feedstream, then diffuses across the membrane and releases the ion into a stripping stream. Regenerated, the extracting agent then shuttles back to the feed side of the membrane to repeat the process. The acidity of the feed and stripping streams is adjusted to favor complexation and decomplexation reactions at the solution-membrane interfaces and to provide a concentration gradient of the coupling ion as a driving force. Typical coupled transport modules are constructed using hollow fibers with diameters on the order of 100 to 1000 .mu.m and are used, for example, to recover metals from plating wastes.
The primary object of this invention is the provision of apparatus and processes for the rapid separation of solutes useful in preparative contexts to resolve complex mixtures including solutions of proteins and isomeric or racemic mixtures, and can be scaled readily to permit any desired throughput. Another object is to provide such apparatus and processes that can be adapted to purify any unique species, provided only that the species is capable of being uniquely reversibly complexed by a proteinaceous binding site.